ABSTRACT Malaria remains one of the most deadly diseases in the world, killing nearly a million people each year. Malaria is hard to control because the immunogenicity of malaria pathogens is very poor, which has made it hard to generate anti-malaria vaccines. The fast spread of insecticide-resistance in mosquito populations and drug- resistance of Plasmodium parasites further serves to increase the rate of malaria transmission. Therefore, there is critical need for the development of novel approaches for malaria control. Since malaria transmission depends on Plasmodium infected mosquitoes, inhibiting parasite infection in mosquitoes represents a novel and practical way to break malaria transmission. At present, most transmission-blocking studies focus on parasite gametocytes in blood with limited success because gametocytes are strongly resistant to drugs. However, very few efforts have been taken to use compounds against mosquito proteins to block malaria transmission. We recently identified the FREP1 gene in wild An. gambiae from malaria endemic areas in Kenya through association studies. Molecular and biochemical analyses revealed that the FREP1 protein mediates the invasion of multiple species of Plasmodium parasites in mosquito midguts through direct interaction with parasites. Based on these findings, we have developed a new high throughput platform to screen a library of natural fungal extracts targeting FREP1, which enabled our team to identify a bioactive compound named P-orlandin that significantly inhibits P. falciparum infection in mosquitoes. Based on these preliminary studies, we hypothesize that small compounds interfere with malaria-mosquito interaction will inhibit malaria transmission. Since multi-pathways involve Plasmodium invasion in mosquitoes, the overarching goal of this application is developing a novel and effective approach for using multiple fungal natural products to block malaria transmission by targeting multiple mosquito proteins that mediate parasites invasion in mosquitoes. We will use our successful collaborative studies as a springboard for identifying additional targets that mediate parasite transmission, as well as small molecules that disrupt the process of malaria transmission. Not only will the compounds we find serve as potential leads for field applications, but they will also serve as essential chemical probes to dissect the molecular biology of the novel pathways we uncover. In this study, we will identify additional candidate genes through genomic-block assistant-associated studies and verify their functional relationship with P. falciparum infection in mosquitoes. The candidate gene products that promote Plasmodium infection in mosquitoes will be chosen as targets to screen for small molecule compounds that block malaria transmission. This work will provide bioactive compounds that are leads for development of drugs or spray reagents to block malaria transmission. In addition, this work provides the malaria communities with new mechanistic insight into Plasmodium transmission to mosquitoes at a molecular level.